Regulatory miPEP Open Reading Frames Contained in the Primary Transcripts of microRNAs.

This review aims to consider retrospectively the available data on the coding properties of pri-microRNAs and the regulatory functions of their open reading frames (ORFs) and the encoded peptides (miPEPs). Studies identifying miPEPs and analyzing the fine molecular mechanisms of their functional activities are reviewed together with a brief description of the methods to identify pri-miRNA ORFs and the encoded protein products. Generally, miPEPs have been identified in many plant species of several families and in a few animal species. Importantly, molecular mechanisms of the miPEP action are often quite different between flowering plants and metazoan species. Requirement for the additional studies in these directions is highlighted by alternative findings concerning negative or positive regulation of pri-miRNA/miRNA expression by miPEPs in plants and animals. Additionally, the question of how miPEPs are distributed in non-flowering plant taxa is very important for understanding the evolutionary origin of such micropeptides. Evidently, further extensive studies are needed to explore the functions of miPEPs and the corresponding ORFs and to understand the full set of their roles in eukaryotic organisms. Thus, we address the most recent integrative views of different genomic, physiological, and molecular aspects concerning the expression of miPEPs and their possible fine functions.

Activity of Chemically Synthesized Peptide Encoded by the miR156A Precursor and Conserved in the Brassicaceae Family Plants.

It was recently found that the primary transcripts of some microRNA genes (pri-miRNAs) are able to express peptides with 12 to 40 residues in length. These peptides, called miPEPs, participate in the transcriptional regulation of their own pri-miRNAs. In our previous studies, we used bioinformatic approach for comparative analysis of pri-miRNA sequences in plant genomes to identify a new group of miPEPs (miPEP-156a peptides) encoded by pri-miR156a in several dozen species of the Brassicaceae family. Exogenous miPEP-156a peptides could efficiently penetrate into the plant seedlings through the root system and spread systemically to the leaves. The peptides produced moderate morphological effect accelerating primary root growth. In parallel, the miPEP-156a peptides upregulated expression of their own pri-miR156a. Importantly, the observed effects at both morphological and molecular levels correlated with the peptide ability to quickly translocate into the cell nucleus and to bind chromatin. In this work, we established secondary structure of the miPEP-156a and demonstrated its changes induced by formation of the peptide complex with DNA.

Probing Structure and Function of Alkali Sensor IRR with Monoclonal Antibodies.

To study the structure and function of the pH-regulated receptor tyrosine kinase insulin receptor-related receptor (IRR), а member of the insulin receptor family, we obtained six mouse monoclonal antibodies against the recombinant IRR ectodomain. These antibodies were characterized in experiments with exogenously expressed full-length IRR by Western blotting, immunoprecipitation, and immunocytochemistry analyses. Utilizing a previously obtained set of IRR/IR chimeras with swapped small structural domains and point amino acid substitutions, we mapped the binding sites of the obtained antibodies in IRR. Five of them showed specific binding to different IRR domains in the extracellular region, while one failed to react with the full-length receptor. Unexpectedly, we found that 4D5 antibody can activate IRR at neutral pH, and 4C2 antibody can inhibit activation of IRR by alkali. Our study is the first description of the instruments of protein nature that can regulate activity of the orphan receptor IRR and confirms that alkali-induced activation is an intrinsic property of this receptor tyrosine kinase.

TAS3 miR390-dependent loci in non-vascular land plants: towards a comprehensive reconstruction of the gene evolutionary history.

Trans-acting small interfering RNAs (ta-siRNAs) are transcribed from protein non-coding genomic TAS loci and belong to a plant-specific class of endogenous small RNAs. These siRNAs have been found to regulate gene expression in most taxa including seed plants, gymnosperms, ferns and mosses. In this study, bioinformatic and experimental PCR-based approaches were used as tools to analyze TAS3 and TAS6 loci in transcriptomes and genomic DNAs from representatives of evolutionary distant non-vascular plant taxa such as Bryophyta, Marchantiophyta and Anthocerotophyta. We revealed previously undiscovered TAS3 loci in plant classes Sphagnopsida and Anthocerotopsida, as well as TAS6 loci in Bryophyta classes Tetraphidiopsida, Polytrichopsida, Andreaeopsida and Takakiopsida. These data further unveil the evolutionary pathway of the miR390-dependent TAS3 loci in land plants. We also identified charophyte alga sequences coding for SUPPRESSOR OF GENE SILENCING 3 (SGS3), which is required for generation of ta-siRNAs in plants, and hypothesized that the appearance of TAS3-related sequences could take place at a very early step in evolutionary transition from charophyte algae to an earliest common ancestor of land plants.

Structural evolution of the 4/1 genes and proteins in non-vascular and lower vascular plants.

The 4/1 protein of unknown function is encoded by a single-copy gene in most higher plants. The 4/1 protein of Nicotiana tabacum (Nt-4/1 protein) has been shown to be alpha-helical and predominantly expressed in conductive tissues. Here, we report the analysis of 4/1 genes and the encoded proteins of lower land plants. Sequences of a number of 4/1 genes from liverworts, lycophytes, ferns and gymnosperms were determined and analyzed together with sequences available in databases. Most of the vascular plants were found to encode Magnoliophyta-like 4/1 proteins exhibiting previously described gene structure and protein properties. Identification of the 4/1-like proteins in hornworts, liverworts and charophyte algae (sister lineage to all land plants) but not in mosses suggests that 4/1 proteins are likely important for plant development but not required for a primary metabolic function of plant cell.

Orthologues of a plant-specific At-4/1 gene in the genus Nicotiana and the structural properties of bacterially expressed 4/1 protein.

Arabidopsis thaliana At-4/1 is the protein of unknown function capable of polar localization in plant cells and intercellular trafficking. In this work, we cloned cDNAs and chromosomal genes of At-4/1 orthologues from several Nicotiana species. Similarly to the 4/1 genes of A. thaliana and Oryza sativa, Nicotiana 4/1 genes have eight exons and seven introns but are considerably longer due to their larger introns. The allotetraploid genome of Nicotiana tabacum, which is known to consist of the 'S genome' originated from Nicotiana sylvestris and the 'T genome' derived from Nicotiana tomentosiformis, encodes two 4/1 genes. The T genome-encoded 4/1 gene, but not that of the S genome, contains a SINE-like transposable element in its intron 2. The 4/1 genes of Nicotiana hesperis and Nicotiana benthamiana lack such an element in the intron 2, but possess a related SINE-like sequence in their intron 4. Collectively, the sequence analysis data provide an insight into the organization of 4/1 genes in flowering plants and the patterns of evolution in the genus Nicotiana. The Nicotiana 4/1 proteins and those of other flowering plants show a significant level of sequence similarity. Computer-assisted analysis was further used to compare their predicted secondary structures. Several algorithms confidently predicted the presence of several coiled-coil domains occupying similar positions in different 4/1 proteins. Analysis of circular dichroism spectra carried out for bacterially expressed N. tabacum 4/1 protein (Nt-4/1) and its N- and C-terminally truncated mutants confirmed that the secondary structure of Nt-4/1 is generally alpha-helical. The C-terminal region of Nt-4/1 was found to undergo a partial proteolysis in Escherichia coli cells. Differential scanning calorimetry of Nt-4/1 protein and its mutants revealed three calorimetric domains most probably corresponding to the N-terminal, central, and C-terminal structural domains of the protein.

Formation of protein complexes containing plant virus movement protein TGBp3 is necessary for its intracellular trafficking.

Cell-to-cell movement of Poa semilatent virus (genus Hordeivirus) in infected plants is mediated by three viral 'triple gene block' (TGB) proteins. One of those termed TGBp3 is an integral membrane protein essential for intracellular transport of other TGB proteins and viral genomic RNA to plasmodesmata. TGBp3 targeting to plasmodesmata-associated sites is believed to involve an unconventional mechanism which does not employ endoplasmic reticulum-derived transport vesicles. Previously TGBp3 has been shown to contain a composite transport signal consisting of the central hydrophilic protein region which includes a conserved pentapeptide YQDLN and the C-terminal transmembrane segment. This study demonstrates that these TGBp3 structural elements have distinct functions in protein transport. The YQDLN-containing region is essential for TGBp3 incorporation into high-molecular-mass protein complexes. In transient expression assay formation of such complexes is necessary for entering the TGBp3-specific pathway of intracellular transport and protein delivery to plasmodesmata-associated sites. In virus-infected plants TGBp3 is also found predominantly in the form of high-molecular-mass complexes. When the complex-formation function of YQDLN-containing region is disabled by a mutation, targeting to plasmodesmata-associated sites can be complemented by a heterologous peptide capable of formation multimeric complexes. The C-terminal transmembrane segment is found to be an essential signal of TGBp3 intracellular transport to peripheral sites.